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Plasmid Replication Initiator Interactions with Origin 13-Mers And Plasmid replication initiator interactions with origin PNAS PLUS 13-mers and polymerase subunits contribute to strand-specific replisome assembly Aleksandra Wawrzycka, Marta Gross, Anna Wasaznik, and Igor Konieczny1 Intercollegiate Faculty of Biotechnology of University of Gdansk and Medical University of Gdansk, 80-822 Gdansk, Poland Edited by Charles C. Richardson, Harvard Medical School, Boston, MA, and approved June 23, 2015 (received for review March 11, 2015) Although the molecular basis for replisome activity has been around the DNA with the use of the clamp loader (reviewed in extensively investigated, it is not clear what the exact mechanism ref. 18). The scenario for the polymerase assembly at the repli- for de novo assembly of the replication complex at the replication cation origin is mainly assumed, based on investigations of the origin is, or how the directionality of replication is determined. mechanism of leading and lagging DNA strand synthesis, con- Here, using the plasmid RK2 replicon, we analyze the protein in- ducted with in vitro assays on primed circular DNA but not on teractions required for Escherichia coli polymerase III (Pol III) ho- supercoiled templates. It is not clear how the replisome is as- loenzyme association at the replication origin. Our investigations sembled on supercoiled dsDNA after origin opening and revealed that in E. coli, replisome formation at the plasmid origin whether the helicase interactions with primase and τ-subunit are involves interactions of the RK2 plasmid replication initiation pro- the only factors contributing to de novo replisome assembly at tein (TrfA) with both the polymerase β-andα-subunits. In the the replication origin. presence of other replication proteins, including DnaA, helicase, In case of bacterial plasmids, involvement of both the plasmid- primase and the clamp loader, TrfA interaction with the β-clamp encoded Rep and the host-encoded replication initiator DnaA contributes to the formation of the β-clamp nucleoprotein complex was reported as essential for origin opening and helicase com- on origin DNA. By reconstituting in vitro the replication reaction on plex recruitment (19–21). DNA replication of the broad-host- ssDNA templates, we demonstrate that TrfA interaction with the range plasmid RK2 (reviewed in ref. 19) is initiated by the RK2 BIOCHEMISTRY β-clamp and sequence-specific TrfA interaction with one strand of plasmid encoded Rep protein (TrfA), which binds to direct re- the plasmid origin DNA unwinding element (DUE) contribute to peats (iterons) localized at the plasmid’s replication origin (oriV) strand-specific replisome assembly. Wild-type TrfA, but not the (22) (Fig. 1A). In contrast to DnaA (23), TrfA, as well as other TrfA QLSLF mutant (which does not interact with the β-clamp), in plasmid Reps, does not contain a DNA binding domain (DBD). the presence of primase, helicase, Pol III core, clamp loader, and Instead, the plasmid Reps are similar to eukaryotic replication β-clamp initiates DNA synthesis on ssDNA template containing initiators and contain a winged helix (WH) domain for DNA 13-mers of the bottom strand, but not the top strand, of DUE. Re- interaction (24, reviewed in 25). TrfA interaction with the iterons sults presented in this work uncovered requirements for anchoring leads to origin opening assisted by host HU and DnaA proteins polymerase at the plasmid replication origin and bring insights of (26). TrfA plays a crucial role in DnaB helicase recruitment and how the directionality of DNA replication is determined. positioning at the AT-rich region of the oriV (27, 28). In contrast to DnaA protein, no data for plasmid Rep filament formation on DNA replication initiation | polymerase III | β-clamp | Rep | plasmid RK2 ssDNA have been provided to date. Recently, it was shown that TrfA interacts with ssDNA containing 13-mer sequences of one NA synthesis of prokaryotic and eukaryotic replicons re- Dquires the coordinated action of several enzymes (reviewed Significance in detail in 1, 2). These enzymes cooperate to form specific nu- cleoprotein complexes during the course of DNA replication. Research on DNA replication initiation has not revealed the ex- The formation of the initial complex is a result of a replication act mechanism for replication complex de novo assembly at the initiation protein (Rep) or origin recognition complex binding to ori origin or how the directionality of replication is determined. To dsDNA within the origin of DNA replication initiation ( ). This date, no evidence for direct involvement of a replication initia- interaction of the replication initiators with DNA results in ori- tion protein (Rep) in the process of polymerase recruitment has gin opening [i.e., destabilization of the DNA unwinding element been reported. This work demonstrates that a plasmid Rep, in (DUE)]. Origin opening provides ssDNA for helicase (3, 4), addition to its already described functions in origin opening and primase (5), and polymerase. helicase recruitment, can serve as a DNA polymerase anchoring It has been demonstrated that during the opening of the oriC factor. Through its interaction with 13-mer sequences on one bacterial chromosomal origin ( ), the chromosomal replica- strand of initially unwound DNA and interactions with the tion initiator, DnaA, binds to specific sequences (DnaA boxes) subunits of DNA polymerase, the initiation protein facilitates (6) and forms a filament on ssDNA (7, 8). Specific interaction strand-specific replisome assembly at the replication origin. This between DnaA and the DnaB helicase (9, 10) recruits the heli- step determines the direction of DNA replication. case and contributes to its loading by a helicase loader, the DnaC τ protein (11). Interactions between DnaB and the -subunit of Author contributions: I.K. designed research; A. Wawrzycka, M.G., and A. Wasaznik per- polymerase (12), as well as DnaB and primase (13), contribute to formed research; A. Wawrzycka, M.G., and I.K. analyzed data; and A. Wawrzycka and I.K. replisome assembly at Escherichia coli oriC. The primase re- wrote the paper. quires contact with single-stranded DNA-binding protein (SSB) The authors declare no conflict of interest. (14) to remain bound to the RNA primer. Disruption of this This article is a PNAS Direct Submission. interaction mediated by the polymerase clamp loader leads to Freely available online through the PNAS open access option. β primase displacement (14); -clamp loading on primed DNA 1To whom correspondence should be addressed. Email: [email protected]. (15, 16); and, finally, interaction of the polymerase core subunits edu.pl. β – β with the -clamp loaded template (14, 17). -clamp loading is a This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. complex reaction involving clamp opening and then positioning 1073/pnas.1504926112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1504926112 PNAS Early Edition | 1of9 Downloaded by guest on September 25, 2021 changed into Ala). Wild-type TrfA (wt TrfA) and TrfA variants A with alterations within the QLSLF motif were purified by affinity chromatography (Materials and Methods). To assess the quality of the purified proteins, we performed an analysis of the iso- thermal CD spectra (Fig. S1) and calculated the content of the respective secondary structures for each TrfA variant. The re- sults did not reveal any substantial differences in the secondary BCstructure’s content of the analyzed TrfA variants in comparison to the wt TrfA. We then determined how the TrfA variants interacted with the β-clamp using surface plasmon resonance (SPR) (SI Materials and Methods and Fig. S2). The wt TrfA immobilized on a CM5 sensor chip interacted with the β-clamp, whereas TrfA ΔLF–β-clamp complex formation was severely impaired. Under the same experimental conditions, TrfA F138A interacted with the β-clamp, although slightly less efficiently than was observed for the wt TrfA. To determine whether the mutations in the QLSLF motif that altered TrfA’s interaction with the β-clamp influenced RK2 DNA synthesis, we tested the replication activity of the purified TrfA variants in an in vitro replication assay. The test was based on E. coli cell crude extract (FII) that allows replication of D supercoiled dsDNA in the presence of the plasmid replication initiator, TrfA. The soluble FII extract contains all proteins necessary for plasmid DNA synthesis, including polymerase III (Pol III) holoenzyme and chaperones for TrfA activation. DNA synthesis in this assay, measured as the total amount of in- corporated nucleotides, reached a maximum when 90 nM wt TrfA was added to the assay mix and was inhibited by larger amounts of this protein (Fig. 1B). Based on the amount of nu- cleotides incorporated into the template, we calculated that 25% of DNA templates were typically copied, similar to results pre- Δ Fig. 1. TrfA LF is not active in RK2 DNA replication either in vitro or in sented by others during experiments with an oriC in vitro system vivo. (A) Plasmid RK2 minimal origin of replication. The scheme presents the (31, 32). TrfA ΔLF was defective in DNA synthesis; replication RK2 origin (oriV) region comprising the cluster of 17-bp direct repeats (iterons), four DnaA boxes, and the DUE region with four 13-mers. (B)In reactions carried out in the presence of varying amounts of this vitro replication with a crude extract (FII) prepared from E. coli C600. (C)In mutant protein remained at background levels. Replication ac- vitro DNA replication reaction reconstituted with purified proteins (Recon. tivity of the TrfA F138A mutant was reduced in comparison to system). (B and C) Both in vitro replication experiments were established wt TrfA but showed a similar activity profile with a peak at with increasing amounts of wt TrfA or TrfA mutants as noted (0, 30, 60, 90, 90 nM protein and an inhibition of DNA synthesis at larger 120, 150, 210, and 300 nM) (results are presented from n = 3 replicates, with amounts of protein.
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